Fluorescence Correlation Spectroscopy as a Versatile Method to Define Aptamer-Protein Interactions with Single-Molecule Sensitivity.

Aptamers are folded oligonucleotides that selectively recognize and bind a target and are consequently regarded as an emerging alternative to antibodies for sensing and therapeutic applications. The rational development of functional aptamers is strictly related to the accurate definition of molecular binding properties. Nevertheless, most of the methodologies employed to define binding affinities use bulk measurements. Here, we describe the use of fluorescence correlation spectroscopy (FCS) as a method with single-molecule sensitivity that quantitatively defines aptamer-protein binding. First, FCS was used to measure the equilibrium affinity between the CLN3 aptamer, conjugated with a dye, and its target, the c-Met protein. Equilibrium affinity was also determined for other functional aptamers targeting nucleolin and platelet-derived growth factors. Then, association and dissociation rates of CLN3 to/from the target protein were measured using FCS by monitoring the equilibration kinetics of the binding reaction in solution. Finally, FCS was exploited to investigate the behavior of CLN3 exposed to physiological concentrations of the most abundant serum proteins. Under these conditions, the aptamer showed negligible interactions with nontarget serum proteins while preserving its affinity for the c-Met. The presented results introduce FCS as an alternative or complementary analytical tool in aptamer research, particularly well-suited for the characterization of protein-targeting aptamers.

A Photoactive Supramolecular Complex Targeting PD-L1 Reveals a Weak Correlation between Photoactivation Efficiency and Receptor Expression Levels in Non-Small-Cell Lung Cancer Tumor Models.

Photo-immunotherapy uses antibodies conjugated to photosensitizers to produce nanostructured constructs endowed with targeting properties and photo-inactivation capabilities towards tumor cells. The superficial receptor density on cancer cells is considered a determining factor for the efficacy of the photodynamic treatment. In this work, we propose the use of a photoactive conjugate that consists of the clinical grade PD-L1-binding monoclonal antibody Atezolizumab, covalently linked to either the well-known photosensitizer eosin or the fluorescent probe Alexa647. Using single-molecule localization microscopy (direct stochastic optical reconstruction microscopy, dSTORM), and an anti-PD-L1 monoclonal antibody labelled with Alexa647, we quantified the density of PD-L1 receptors exposed on the cell surface in two human non-small-cell lung cancer lines (H322 and A549) expressing PD-L1 to a different level. We then investigated if this value correlates with the effectiveness of the photodynamic treatment. The photodynamic treatment of H322 and A549 with the photo-immunoconjugate demonstrated its potential for PDT treatments, but the efficacy did not correlate with the PD-L1 expression levels. Our results provide additional evidence that receptor density does not determine a priori the level of photo-induced cell death.

Nitric Oxide Sensing by a Blue Fluorescent Protein.

S-Nitrosylation of cysteine residues is an important molecular mechanism for dynamic, post-translational regulation of several proteins, providing a ubiquitous redox regulation. Cys residues are present in several fluorescent proteins (FP), including members of the family of Aequorea victoria Green Fluorescent Protein (GFP)-derived FPs, where two highly conserved cysteine residues contribute to a favorable environment for the autocatalytic chromophore formation reaction. The effect of nitric oxide on the fluorescence properties of FPs has not been investigated thus far, despite the tremendous role FPs have played for 25 years as tools in cell biology. We have examined the response to nitric oxide of fluorescence emission by the blue-emitting fluorescent protein mTagBFP2. To our surprise, upon exposure to micromolar concentrations of nitric oxide, we observed a roughly 30% reduction in fluorescence quantum yield and lifetime. Recovery of fluorescence emission is observed after treatment with Na-dithionite. Experiments on related fluorescent proteins from different families show similar nitric oxide sensitivity of their fluorescence. We correlate the effect with S-nitrosylation of Cys residues. Mutation of Cys residues in mTagBFP2 removes its nitric oxide sensitivity. Similarly, fluorescent proteins devoid of Cys residues are insensitive to nitric oxide. We finally show that mTagBFP2 can sense exogenously generated nitric oxide when expressed in a living mammalian cell. We propose mTagBFP2 as the starting point for a new class of genetically encoded nitric oxide sensors based on fluorescence lifetime imaging.

Probing the Role of Murine Neuroglobin CDloop-D-Helix Unit in CO Ligand Binding and Structural Dynamics.

We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop-D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop-D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop-D-helix in neuroprotective signaling in a cellular or animal model.

A red-green photochromic bacterial protein as a new contrast agent for improved photoacoustic imaging.

The GAF3 domain of the cyanobacteriochrome Slr1393 from Synechocystis sp. PCC6803, binding phycocyanobilin as a chromophore, shows photochromicity between two stable, green- and red-absorbing states, characterized by relatively high photoconversion yields. Using nanosecond-pulsed excitation by red or green light, respectively, and suitable cw photoconversion beams, we demonstrate that the light-modulatable photoacoustic waveforms arising from GAF3 can be easily distinguished from background signals originating from non-modulatable competitive absorbers and scattering media. It is demonstrated that this effect can be exploited to identify the position of the photochromic molecule by using as a phantom a cylindrical capillary tube filled with either a GAF3 solution or with an E.coli suspension overexpressing GAF3. These properties identify the high potential of GAF3 to be included in the palette of genetically encoded photochromic probes for photoacoustic imaging.

Versatile Supramolecular Complex for Targeted Antimicrobial Photodynamic Inactivation.

We report the development of a supramolecular structure endowed with photosensitizing properties and targeting capability for antimicrobial photodynamic inactivation. Our synthetic strategy uses the tetrameric bacterial protein streptavidin, labeled with the photosensitizer eosin, as the main building block. Biotinylated immunoglobulin G (IgG) from human serum, known to associate with Staphylococcus aureus protein A, was bound to the complex streptavidin-eosin. Fluorescence correlation spectroscopy and fluorescence microscopy demonstrate binding of the complex to S. aureus. Efficient photoinactivation is observed for S. aureus suspensions treated with IgG-streptavidin-eosin at concentrations higher than 0.5 µM and exposed to green light. The proposed strategy offers a flexible platform for targeting a variety of molecules and microbial species.

A photosensitizing fusion protein with targeting capabilities.

The photodynamic treatment for antimicrobial applications or anticancer therapy relies on reactive oxygen species generated by photosensitizing molecules after absorption of visible or near-infrared light. If the photosensitizing molecule is in close vicinity of the microorganism or the malignant cell, a photocytotoxic action is exerted. Therefore, the effectiveness of photosensitizing compounds strongly depends on their capability to target microbial or cancer-specific proteins. In this study, we report on the preparation and preliminary characterization of human recombinant myoglobin fused to the vasoactive intestinal peptide to target vasoactive intestinal peptide receptor (VPAC) receptors. Fe-protoporphyrin IX was replaced by the photosensitizing compound Zn-protoporphyrin IX. Taking advantage of the fluorescence emission by Zn-protoporphyrin IX, we show that the construct can bind prostate cancer cells where the VPAC receptors are expressed.

Targeted photoimmunotherapy for cancer.

Photodynamic therapy (PDT) is a clinically approved procedure that can exert a curative action against malignant cells. The treatment implies the administration of a photoactive molecular species that, upon absorption of visible or near infrared light, sensitizes the formation of reactive oxygen species. These species are cytotoxic and lead to tumor cell death, damage vasculature, and induce inflammation. Clinical investigations demonstrated that PDT is curative and does not compromise other treatment options. One of the major limitations of the original method was the low selectivity of the photoactive compounds for malignant over healthy tissues. The development of conjugates with antibodies has endowed photosensitizing molecules with targeting capability, so that the compounds are delivered with unprecedented precision to the site of action. Given their fluorescence emission capability, these supramolecular species are intrinsically theranostic agents.

The Interaction of Hypericin with SARS-CoV-2 Reveals a Multimodal Antiviral Activity.

Hypericin is a photosensitizing drug that is active against membrane-enveloped viruses and therefore constitutes a promising candidate for the treatment of SARS-CoV-2 infections. The antiviral efficacy of hypericin is largely determined by its affinity toward viral components and by the number of active molecules loaded on single viruses. Here we use an experimental approach to follow the interaction of hypericin with SARS-CoV-2, and we evaluate its antiviral efficacy, both in the dark and upon photoactivation. Binding to viral particles is directly visualized with fluorescence microscopy, and a strong affinity for the viral particles, most likely for the viral envelope, is measured spectroscopically. The loading of a maximum of approximately 30 molecules per viral particle is estimated, despite with marked heterogeneity among particles. Because of this interaction, nanomolar concentrations of photoactivated hypericin substantially reduce virus infectivity on Vero E6 cells, but a partial effect is also observed in dark conditions, suggesting multiple mechanisms of action for this drug.

A Double Payload Complex between Hypericin and All-trans Retinoic Acid in the ß-Lactoglobulin Protein.

Combined therapies are usually used to treat acne vulgaris since this approach can tackle various foci simultaneously. Using a combination of spectroscopic, computational, and microbiological techniques and methods, herein we report on the use of ß-lactoglobulin as a double payload carrier of hypericin (an antimicrobial photodynamic agent) and all-trans retinoic acid (an anti-inflammatory drug) for S. aureus in vitro photodynamic inactivation. The addition of all-trans retinoic acid to hypericin-ß-lactoglobulin complex renders a photochemically safe vehicle due to the photophysical quenching of hypericin, which recovers its photodynamic activity when in contact with bacteria. The ability of hypericin to photoinactivate S. aureus was not affected by retinoic acid. ß-Lactoglobulin is a novel biocompatible and photochemically safe nanovehicle with strong potential for the treatment of acne.

From hemoglobin allostery to hemoglobin-based oxygen carriers.

Hemoglobin (Hb) plays its vital role through structural and functional properties evolutionarily optimized to work within red blood cells, i.e., the tetrameric assembly, well-defined oxygen affinity, positive cooperativity, and heterotropic allosteric regulation by protons, chloride and 2,3-diphosphoglycerate. Outside red blood cells, the Hb tetramer dissociates into dimers, which exhibit high oxygen affinity and neither cooperativity nor allosteric regulation. They are prone to extravasate, thus scavenging endothelial NO and causing hypertension, and cause nephrotoxicity. In addition, they are more prone to autoxidation, generating radicals. The need to overcome the adverse effects associated with cell-free Hb has always been a major hurdle in the development of substitutes of allogeneic blood transfusions for all clinical situations where blood is unavailable or cannot be used due to, for example, religious objections. This class of therapeutics, indicated as hemoglobin-based oxygen carriers (HBOCs), is formed by genetically and/or chemically modified Hbs. Many efforts were devoted to the exploitation of the wealth of biochemical and biophysical information available on Hb structure, function, and dynamics to design safe HBOCs, overcoming the negative effects of free plasma Hb. Unfortunately, so far, no HBOC has been approved by FDA and EMA, except for compassionate use. However, the unmet clinical needs that triggered intensive investigations more than fifty years ago are still awaiting an answer. Recently, HBOCs "repositioning" has led to their successful application in organ perfusion fluids.

Unusually Fast bis-Histidyl Coordination in a Plant Hemoglobin.

The recently identified nonsymbiotic hemoglobin gene MtGlb1-2 of the legume Medicago truncatula possesses unique properties as it generates four alternative splice forms encoding proteins with one or two heme domains. Here we investigate the ligand binding kinetics of MtGlb1-2.1 and MtGlb1-2.4, bearing two hemes and one heme, respectively. Unexpectedly, the overall time-course of ligand rebinding was unusually fast. Thus, we complemented nanosecond laser flash photolysis kinetics with data collected with a hybrid femtosecond-nanosecond pump-probe setup. Most photodissociated ligands are rebound geminately within a few nanoseconds, which leads to rates of the bimolecular rebinding to pentacoordinate species in the 108 M-1s-1 range. Binding of the distal histidine to the heme competes with CO rebinding with extremely high rates (kh ~ 105 s-1). Histidine dissociation from the heme occurs with comparable rates, thus resulting in moderate equilibrium binding constants (KH ~ 1). The rate constants for ligation and deligation of distal histidine to the heme are the highest reported for any plant or vertebrate globin. The combination of microscopic rates results in unusually high overall ligand binding rate constants, a fact that contributes to explaining at the mechanistic level the extremely high reactivity of these proteins toward the physiological ligands oxygen, nitric oxide and nitrite.

Mycobacterial and Human Ferrous Nitrobindins: Spectroscopic and Reactivity Properties.

Structural and functional properties of ferrous Mycobacterium tuberculosis (Mt-Nb) and human (Hs-Nb) nitrobindins (Nbs) were investigated. At pH 7.0 and 25.0 °C, the unliganded Fe(II) species is penta-coordinated and unlike most other hemoproteins no pH-dependence of its coordination was detected over the pH range between 2.2 and 7.0. Further, despite a very open distal side of the heme pocket (as also indicated by the vanishingly small geminate recombination of CO for both Nbs), which exposes the heme pocket to the bulk solvent, their reactivity toward ligands, such as CO and NO, is significantly slower than in most hemoproteins, envisaging either a proximal barrier for ligand binding and/or crowding of H2O molecules in the distal side of the heme pocket which impairs ligand binding to the heme Fe-atom. On the other hand, liganded species display already at pH 7.0 and 25 °C a severe weakening (in the case of CO) and a cleavage (in the case of NO) of the proximal Fe-His bond, suggesting that the ligand-linked movement of the Fe(II) atom onto the heme plane brings about a marked lengthening of the proximal Fe-imidazole bond, eventually leading to its rupture. This structural evidence is accompanied by a marked enhancement of both ligands dissociation rate constants. As a whole, these data clearly indicate that structural-functional relationships in Nbs strongly differ from what observed in mammalian and truncated hemoproteins, suggesting that Nbs play a functional role clearly distinct from other eukaryotic and prokaryotic hemoproteins.

A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

In plants, symbiotic hemoglobins act as carriers and buffers of O2 in nodules, whereas nonsymbiotic hemoglobins or phytoglobins (Glbs) are ubiquitous in tissues and may perform multiple, but still poorly defined, functions related to O2 and/or nitric oxide (NO). Here, we have identified a Glb gene of the model legume Medicago truncatula with unique properties. The gene, designated MtGlb1-2, generates four alternative splice forms encoding Glbs with one or two heme domains and 215-351 amino acid residues. This is more than double the size of any hemoglobin from plants or other organisms described so far. A combination of molecular, cellular, biochemical, and biophysical methods was used to characterize these novel proteins. RNA-sequencing showed that the four splice variants are expressed in plant tissues. MtGlb1-2 is transcriptionally activated by hypoxia and its expression is further enhanced by an NO source. The gene is preferentially expressed in the meristems and vascular bundles of roots and nodules. Two of the proteins, bearing one or two hemes, were characterized using mutants in the distal histidines of the hemes. The Glbs are extremely reactive toward the physiological ligands O2, NO, and nitrite. They show very high O2 affinities, NO dioxygenase activity (in the presence of O2), and nitrite reductase (NiR) activity (in the absence of O2) compared with the hemoglobins from vertebrates and other plants. We propose that these Glbs act as either NO scavengers or NO producers depending on the O2 tension in the plant tissue, being involved in the fast and fine tuning of NO concentration in the cytosol in response to sudden changes in O2 availability.

Tetramethylbenzidine: An Acoustogenic Photoacoustic Probe for Reactive Oxygen Species Detection.

Photoacoustic imaging is attracting a great deal of interest owing to its distinct advantages over other imaging techniques such as fluorescence or magnetic resonance image. The availability of photoacoustic probes for reactive oxygen and nitrogen species (ROS/RNS) could shed light on a plethora of biological processes mediated by these key intermediates. Tetramethylbenzidine (TMB) is a non-toxic and non-mutagenic colorless dye that develops a distinctive blue color upon oxidation. In this work, we have investigated the potential of TMB as an acoustogenic photoacoustic probe for ROS/RNS. Our results indicate that TMB reacts with hypochlorite, hydrogen peroxide, singlet oxygen, and nitrogen dioxide to produce the blue oxidation product, while ROS, such as the superoxide radical anion, sodium peroxide, hydroxyl radical, or peroxynitrite, yield a colorless oxidation product. TMB does not penetrate the Escherichia coli cytoplasm but is capable of detecting singlet oxygen generated in its outer membrane.

Structural and functional properties of Antarctic fish cytoglobins-1: Cold-reactivity in multi-ligand reactions.

While the functions of the recently discovered cytoglobin, ubiquitously expressed in vertebrate tissues, remain uncertain, Antarctic fish provide unparalleled models to study novel protein traits that may arise from cold adaptation. We report here the spectral, ligand-binding and enzymatic properties (peroxynitrite isomerization, nitrite-reductase activity) of cytoglobin-1 from two Antarctic fish, Chaenocephalus aceratus and Dissostichus mawsoni, and present the crystal structure of D. mawsoni cytoglobin-1. The Antarctic cytoglobins-1 display high O2 affinity, scarcely compatible with an O2-supply role, a slow rate constant for nitrite-reductase activity, and do not catalyze peroxynitrite isomerization. Compared with mesophilic orthologues, the cold-adapted cytoglobins favor binding of exogenous ligands to the hexa-coordinated bis-histidyl species, a trait related to their higher rate constant for distal-His/heme-Fe dissociation relative to human cytoglobin. At the light of a remarkable 3D-structure conservation, the observed differences in ligand-binding kinetics may reflect Antarctic fish cytoglobin-1 specific features in the dynamics of the heme distal region and of protein matrix cavities, suggesting adaptation to functional requirements posed by the cold environment. Taken together, the biochemical and biophysical data presented suggest that in Antarctic fish, as in humans, cytoglobin-1 unlikely plays a role in O2 transport, rather it may be involved in processes such as NO detoxification.

Mycobacterial and Human Nitrobindins: Structure and Function.

Aims: Nitrobindins (Nbs) are evolutionary conserved all-ß-barrel heme-proteins displaying a highly solvent-exposed heme-Fe(III) atom. The physiological role(s) of Nbs is almost unknown. Here, the structural and functional properties of ferric Mycobacterium tuberculosis Nb (Mt-Nb(III)) and ferric Homo sapiens Nb (Hs-Nb(III)) have been investigated and compared with those of ferric Arabidopsis thaliana Nb (At-Nb(III), Rhodnius prolixus nitrophorins (Rp-NP(III)s), and mammalian myoglobins. Results: Data here reported demonstrate that Mt-Nb(III), At-Nb(III), and Hs-Nb(III) share with Rp-NP(III)s the capability to bind selectively nitric oxide, but display a very low reactivity, if any, toward histamine. Data obtained overexpressing Hs-Nb in human embryonic kidney 293 cells indicate that Hs-Nb localizes mainly in the cytoplasm and partially in the nucleus, thanks to a nuclear localization sequence encompassing residues Glu124-Leu154. Human Hs-Nb corresponds to the C-terminal domain of the human nuclear protein THAP4 suggesting that Nb may act as a sensor possibly modulating the THAP4 transcriptional activity residing in the N-terminal region. Finally, we provide strong evidence that both Mt-Nb(III) and Hs-Nb(III) are able to scavenge peroxynitrite and to protect free l-tyrosine against peroxynitrite-mediated nitration. Innovation: Data here reported suggest an evolutionarily conserved function of Nbs related to their role as nitric oxide sensors and components of antioxidant systems. Conclusion: Human THAP4 may act as a sensing protein that couples the heme-based Nb(III) reactivity with gene transcription. Mt-Nb(III) seems to be part of the pool of proteins required to scavenge reactive nitrogen and oxygen species produced by the host during the immunity response.

Photodynamic action of Hypericum perforatum hydrophilic extract against Staphylococcus aureus.

Hypericin (Hyp) is one of the most effective, naturally occurring photodynamic agents, which proved effective against a wide array of microorganisms. One limitation of its large scale application as a disinfectant is the high production cost of the pure compound. The availability of photoactive materials at a lower cost may be highly beneficial to the actual implementation of photodisinfection also at the industrial level. In this work we report the use of a lyophilized extract from Hypericum perforatum as a photosensitizing material. We show that optical absorption in the green-red region of the visible spectrum of ethanol or DMSO solutions of the lyophilized extract contains bands arising from Hyp. When excited with light in the main Hyp absorption bands, fluorescence emission and triplet state formation occur as in pure Hyp solutions. We show that ethanol or DMSO solutions of the lyophilized extract from Hypericum perforatum are highly efficient photodynamic agents against Gram-positive Staphylococcus aureus, chosen as a model. The performance is indistinguishable from that of the pure compound. Using fluorescence microscopy, we demonstrate that upon incubation of S. aureus with lyophilized extract solutions, Hyp is found on the bacterial wall, as previously reported for the pure compound.

Corrigendum: A Plant Gene Encoding One-Heme and Two-Heme Hemoglobins With Extreme Reactivities Toward Diatomic Gases and Nitrite.

[This corrects the article DOI: 10.3389/fpls.2020.600336.].

Dynamics and efficiency of photoswitching in biliverdin-binding phytochromes.

The light-driven conversions between the dark-adapted and the photoproduct state were recorded for bacteriophytochromes (BphP) carrying biliverdin IXα (BV) as chromophore by time-resolved absorption spectroscopy. BphPs can be photoswitched between a red absorbing (Pr, maximum at ca. 700 nm) and a far-red/near-infrared (Pfr, maximum at ca. 750 nm) absorbing state, thereby showing a considerable red-shift with respect to plant phytochromes. Representatives for BphPs studied here are: PstBphP1 from Pseudomonas syringae pv. tomato, for which Pfr is the photoproduct; the bathy-phytochrome PaBphP from Pseudomonas aeruginosa for which instead Pfr is the thermally stable parental state. The third BphP-like protein was FphA from the fungus Aspergillus nidulans, a eukaryotic protein also carrying BV as a chromophore, for which Pr is considered to be the dark-adapted state. All three BphPs show a canonical modular arrangement with a three-domain photosensory module (PAS-GAF-PHY) and a histidine-kinase (HK) signalling domain. The quantum yields for Pr-to-Pfr photoconversion are in the range 0.02-0.12, and 0.04-0.08 for the Pfr-to-Pr route. Photoproducts of both bacterial phytochromes thermally recovered in the dark, whereas for the fungal protein (FphA) both Pr and Pfr forms are thermally stable for days and could be interconverted only by selective irradiation. The photoinduced reactions of all three BV-phytochromes are in general kinetically less complex than those of plant phytochromes, with the notable exception of the Pr-to-Pfr route for PstBphP1. By contrast in the Pfr-to-Pr conversion of FphAN753 the final product is already formed during the very early steps of the process, without formation of any further intermediates: to our knowledge it is the first phytochrome showing this behavior. All three proteins investigated are weakly fluorescent in the Pr form, with a maximum fluorescence quantum yield of 0.02 (PaBphP), and have undetectable fluorescence in the Pfr state.